Sensitive detection of Listeria monocytogenes based on highly efficient enrichment with vancomycin-conjugated brush-like magnetic nano-platforms.

Pathogens pose a significant threat to public health worldwide. Despite many technological advances in the rapid diagnosis of pathogens, sensitive pathogen detection remains challenging because target pathogenic bacteria usually exist in complex samples at very low concentrations. Here, the construction of multivalent brush-like magnetic nanoprobes and their application for the efficient enriching of pathogens are demonstrated. Brush-like magnetic nanoprobes were constructed by modification with poly-L-lysine (PLL) onto amino-modified magnetic beads, followed by coupling of PEG (amine-PEG5000-COOH) to the amine sites of PLL. Subsequently, vancomycin (Van), a small-molecule antibiotic with affinity to the terminal peptide (D-alanyl-D-alanine) on the cell wall of Gram-positive bacteria, was conjugated to the carboxyl of the PEG. The use of multivalent brush-like magnetic nanoprobes (Van-PEG-PLL-MNPs) results in a high enrichment efficiency (>94%) and satisfactory purity for Listeria monocytogenes (employed as a model) within 20min, even at bacterial concentrations of only 102cfumL-1. Integrated with the enrichment of the Van-PEG-PLL-MNP nano-platform and electrochemiluminescence (ECL) detection, Listeria monocytogenes can be rapidly and accurately detected at levels as low as 10cfumL-1. The approach described herein holds great potential for realizing rapid and sensitive pathogen detection in clinical samples.

Invading stacking primer: A trigger for high-efficiency isothermal amplification reaction with superior selectivity for detecting microRNA variants.

Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as "invading stacking primer (IS-Primer)" which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays.

Construction of Fe3O4/Vancomycin/PEG Magnetic Nanocarrier for Highly Efficient Pathogen Enrichment and Gene Sensing.

Infectious diseases, especially pathogenic bacterial infections, pose a growing threat to public health worldwide. As pathogenic bacteria usually exist in complex experimental matrixes at very low concentrations, developing a technology for rapid and biocompatible sample enrichment is essential for sensitive diagnosis. In this study, an Fe3O4/Vancomycin/PEG magnetic nanocarrier was constructed for efficient sample enrichment and in situ nucleic acid preparation of pathogenic bacteria for subsequent gene sensing. We attached Vancomycin, a well-known broad-spectrum antibiotic, to the surface of Fe3O4 nanoparticles as a universal molecular probe to target bacterial cells. Polyethylene glycol (PEG) was introduced to enhance the nanocarrier's water solubility and biocompatibility. Results show that the proposed nanocarrier achieved a 90% capture efficiency even if at a Listeria monocytogenes concentration of 1×10(2) cfu/mL. Contributing to the good water solubility achieved by the employment of modified PEG, highly efficient enrichment (enrichment factor 10 times higher than PEG-free nanocarrier) can be completed in 30 min. Moreover, PEG would also develop the nanoparticles' biocompatibility by passivating the positively charged unreacted amines on the magnetic nanoparticles, thus helping to release the negatively charged bacterial genome from the nanocarrier/bacteria complexes when an in situ nucleic acids extraction step was executed. The outstanding bacterial capture capability and biocompatibility of this nanocarrier enabled the implementation of a highly sensitive gene-sensing strategy of pathogens. By employing an electrochemiluminescence-based gene-sensing assay, L. monocytogenes can be rapidly detected with a limit of detection of 10 cfu/mL, which shows great potential for clinical applications.

Miniaturized paper-based gene sensor for rapid and sensitive identification of contagious plant virus.

Plant viruses cause significant production and economic losses in the agricultural industry worldwide. Rapid and early identification of contagious plant viruses is an essential prerequisite for the effective control of further spreading of infection. In this work, we describe a miniaturized paper-based gene sensor for the rapid and sensitive identification of a contagious plant virus. Our approach makes use of hybridization-mediated target capture based on a miniaturized lateral flow platform and gold nanoparticle colorimetric probes. The captured colorimetric probes on the test line and control line of the gene sensor produce characteristic red bands, enabling visual detection of the amplified products within minutes without the need for sophisticated instruments or the multiple incubation and washing steps performed in most other assays. Quantitative analysis is realized by recording the optical intensity of the test line. The sensor was used successfully for the identification of banana bunchy top virus (BBTV). The detection limit was 0.13 aM of gene segment, which is 10 times higher than that of electrophoresis and provides confirmation of the amplified products. We believe that this simple, rapid, and sensitive bioactive platform has great promise for warning against plant diseases in agricultural production.

Visual and sensitive detection of viable pathogenic bacteria by sensing of RNA markers in gold nanoparticles based paper platform.

Food-borne pathogens have been recognized as a major cause of human infections worldwide. Their identification needs to be simpler, cheaper and more reliable than the traditional methods. Here, we constructed a low-cost paper platform for viable pathogenic bacteria detection with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test using sandwich hybridization assays. When the RNA products migrated along the platform by capillary action, the gold nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 0.5 pg/µL genomic RNA from L. monocytogenes could be detected. It could also be used to specifically detect 20 CFU/mL L. monocytogenes from actual samples. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens, as it can overcome the false-positive results caused by amplifying nonviable L. monocytogenes. Furthermore, the results can be imaged and transformed into a two-dimensional bar code through an Android-based smart phone for further analysis or in-field food safety tracking.

[The features of four types of operation for treating chronic hypertrophy rhinitis and ultrastructure observation of the inferior turbinate mucosa].

OBJECTIVE: To assess the features of four types of operation for treating chronic hypertrophy rhinitis and to observe the ultrastructure of inferior turbinate mucosa. METHOD: Eighty-eight cases of chronic hypertrophic rhinitis (HR) were treated by transnasal endoscopic submucous inferior turbinate resection (group A), thirty cases of HR were treated by partial inferior turbinectomy (group B), thirty six cases of HR were treated by bipolar radiofrequency ablation on inferior turbinate (group C), ten cases of HR were treated by injection of sclerosing agent (group D). The efficacy in the three groups (A,B,C) and the features of four types operation were compared and the ultrastructure of inferior turbinate was observed both preoperatively and postoperatively. RESULT: Four groups of HR were followed up 4 months to half year after operation, effective rate was 97.4 (group A) 100% (group B) and 93.4% (group C) respectively, while group A, group B and group C effective rate had no significant difference among them. Group A and group C have more advantages compared to the other 2 groups for they remained good ultrastructure of inferior turbinate mucosal cilia after 4 to 6 months postoperatively. CONCLUSIONS: The use of transnasal endoscopic submucous inferior turbinate resection and bipolar radiofrequency ablation for treatment of chronic hypertrophy rhinitis are effective modality for the treatment of chronic hypertrophy rhinitis.